Optogenetics refers to the use of light in conjunction with light-sensitive proteins to stimulate excitable cells and tissues. This project aims at extending the utility of optogenetics to cardiac applications. We exploit the heart's efficient cell to cell coupling, to develop the first cell delivery system for expression of light-sensitive ion channels, LSICs, i.e. non-viral cardiac optogenetics. We term our strategy a tandem cell unit (TCU) approach, where a light-responsive unit is formed by a host cardiomyocyte and a non-excitable donor cell carrying exogenous LSICs. Biophysically, for this unit to function (to fire an action potential upon light excitation), low-resistance coupling is required. Although in its infancy, cardiac optogenetics can be demonstrated effective for cardiac pacing, with a therapeutic application in the clinic, and as a research tool ideal for dissection of cardiac arrhythmias in vitro and perhaps in vivo owing to the robustness, remote nature and spatiotemporal precision of optical control. The driving hypothesis of this proposal is that optogenetics can provide a useful research and clinical approach to study cardiac excitability. Our experimental aims to test this hypothesis will employ single cardiac myocytes, tandem cell units and cardiac syncytia in vitro, along with in silico simulations and ultimately combined in vivo/in vitro proof of principle experiments for whole heart applications. The Specific Aims are as follows: 1) Conduct comprehensive biophysical characterization of LSICs and develop in silico tools for cardiac optogenetics and integrate those in a dynamic clamp; 2) Quantify energy requirements of LSIC-based control of cardiac excitation and demonstrate optimization strategies for cardiac optogenetics based on the TCU concept; 3) Demonstrate utility of cardiac optogenetics in pacing and cardioversion in vitro; 4) Devise proof of principle experiments for in vivo optical pacing based on optimization results: If successful, we will determine the optimal donor cell parameters to minimize energy consumption and maximize spatial control of light-induced cardiac excitability and refractoriness in vitro, in silico and ex vivo at the single cell, tandem cell unit, cardiac syncytium and whole heart level. These experiments serve as a necessary prelude to therapeutic applications of optogenetics to the heart. PUBLIC HEALTH RELEVANCE: This project offers several important innovations: 1) pioneering work in extending the use of optogenetics to control of cardiac muscle; 2) validation of a new approach to optogenetics - a non-viral cell delivery with translational potential for cardiac pacing; 3) quantitative biophysical analysis of light-sensitive ion channels and development of mathematical and experimental tools to facilitate optogenetic investigation and optimize optical stimulation beyond the cardiac field.